Identification of genes altered in multiple myeloma

ABSTRACT

This invention provides a method of determining a chromosomal breakpoint in a subject suffering from multiple myeloma which comprises steps of: (a) obtaining a DNA sample from the subject suffering from multiple myeloma; (b) determining whether there is J and C disjunction in the immunoglobulin heavy chain gene in the obtained DNA sample; (c) obtaining a genomic library having clones which contain genomic DNA fragments from the DNA sample which shows positive J and C disjunction; (d) selecting and isolating clones of the obtained library which show positive hybridization with a probe which is capable of specifically hybridizing with the C but not the J region of the immunoglobulin heavy chain gene; (e) preparing fluorescent probes from the genomic DNA fragments of the isolated clones from step (d); (f) hybridizing said fluorescent probes with metaphase chromosomes; and (g) determining the identity of the chromosomes which are capable of hybridizing to said fluorescent probes, wherein the identification of a chromosome other than chromosome 14 would indicate that the chromosomal breakpoint is between chromosome 14 and the identified chromosome, thereby determining a chromosomal breakpoint in a subject suffering from multiple myeloma. This invention also provides the identified gene altered by a chromosomal breakpoint and various uses thereof.

The invention disclosed herein was made with Government support under NIH Grant No. CA 44025. Accordingly, the U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.

Multiple myeloma (MM) is an incurable B cell tumor affecting B cell end-stage differentiation. Clinically, the course of MM is similar to end-stage plasma cell leukemia (PCL), i.e., there is an uncontrollable proliferation of myeloma cells accompanied by numerous complications, including hyperviscosity syndromes, hypercalcemia, infections, multiple bone fractures, and organ failure.

Non-random chromosomal translocation is known to play a crucial role in the tumorigenesis of hematologic malignancies (1). In B-cell lymphomas, many important proto-oncogenes deregulated by juxtaposition to immunoglobulin (Ig) gene locus have been identified. Each proto-oncogene is associated with a specific subtype of lymphoma, such as c-MYC in Burkitt's lymphoma, Cyclin DI IBCLI in mantle cell lymphoma, BCL-2 in follicular lymphoma and BCL-6 in diffuse large cell lymphoma (2-8). In contrast, little is known about molecular alterations of human MM/PCL, due to the difficulty in cytogenetic analysis. However, previous cytogenetic reports have shown a 14q+ chromosome, suggesting the existence of a chromosomal translocation involving the Ig heavy chain (IgH) locus, is observed in 20˜30% of the MM/PCL cases and it is the most frequent consistent abnormality (9-12). Even in such cases, most cytogenetic data have failed to identify donor chromosomes other than 11q13, 8q24, and 18q21, where proto-oncogenes Cyclin DIIBCL-IIPRADI, c-MYC and BCL-2 are located, respectively. Among them, the 11q13 locus has been demonstrated to be involved in nearly 5˜10% of the cases and also in 62% of the established cell lines (13). The t(11;14) (q13;q32) translocation is also accompanied by a corresponding overexpression of the Cyclin Dl gene, which raises a strong possibility of the involvement of this gene, although the breakpoints at 11q13 do not cluster like those of the lymphoma cases (14-16). Recent advances in fluorescence in situ hybridization (FISH) have made it possible to clarify both the frequency of the 14q+ chromosomes and the partner chromosomes of the IgH loci. One such report revealed an intriguing result, i.e., that numerous chromosomal loci are able to translocate to IgH locus, including 6p21, 1q21, 3p11, 7q11, 11q23 (17). This has prompted a search for the proto-oncogenes deregulated by the regulatory elements of the IgH gene for a further understanding of the molecular mechanisms of MM/PCL. In the present study, one candidate proto-oncogene, MUM1 (multiple myeloma oncogene 1), was found juxtaposed to the IgH gene as a result of t(6;14)(p25; q32) translocation in human myeloma cell line, SKMM-1. Over expression of the MUM1 mRNA was observed in this cell line. A second gene, called MUM-2 was found translocated in proximity to the IgH gene on chromosome 14q32 in human myeloma cell line, U-266.

The method of analysis of 14q+ chromosomal translocations and identification of the genes altered in multiple myeloma of this invention are useful since 1) no method is currently available to determine the chromosomal sequences involved in 14q+ translocations, the most important cytogenetic lesions associated with MM pathogenesis; 2) no specific gene lesion is currently known for MM; 3) no diagnostic method based on gene/DNA lesion is currently available for MM and 4) there are no therapeutic approaches aimed at counteracting the action of abnormal gene products in MM.

SUMMARY OF THE INVENTION

This invention provides a method of determining a chromosomal breakpoint in a subject suffering from multiple myeloma which comprises steps of: (a) obtaining a DNA sample from the subject suffering from multiple myeloma; (b) determining whether there is J and C disjunction in the immunoglobulin heavy chain gene in the obtained DNA sample; (c) obtaining a genomic library having clones which contain genomic DNA fragments from the DNA sample which shows positive J and C disjunction; (d) selecting and isolating clones of the obtained library which show positive hybridization with a probe which is capable of specifically hybridizing with the C but not the J region of the immunoglobulin heavy chain gene; (e) preparing fluorescent probes from the genomic DNA fragments of the isolated clones from step (d); (f) hybridizing said fluorescent probes with metaphase chromosomes; and (g) determining the identity of the chromosomes which are capable of hybridizing to said fluorescent probes, wherein the identification of a chromosome other than chromosome 14 would indicate that the chromosomal breakpoint is between chromosome 14 and the identified chromosome, thereby determining a chromosomal breakpoint in a subject suffering from multiple myeloma.

This invention provides a method to identify a gene other than the immunoglobulin gene which is located in chromosome 14, altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma which comprises steps of: a) selecting a probe having a sequence of a chromosome other than chromosome 14, identified at the chromosomal breakpoint detected in a subject suffering fron multiple myeloma, wherein said probe is capable of hybridizing to the unique sequence of the gene other than the immunoglobulin gene altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma; b) contacting said probe with mRNA isolated from a cell under conditions permitting formation of a complex between said probe and the mRNA; c) isolating the complex resulting from step (b); d) determining the sequence of the mRNA in the isolated complex, thereby determining the identity of the gene.

This invention provides a gene designated MUM-1. This invention provides a gene designated MUM-2. This invention provides an isolated nucleic acid molecule encoding a MUM protein. This invention provides a DNA encoding a MUM protein. This invention provides a cDNA encoding a MUM protein. This invention provides a genomic DNA molecule encoding a MUM protein. This invention provides a RNA molecule encoding a MUM protein. This invention provides an isolated nucleic acid molecule encoding a human MUM-1 protein. This invention provides an isolated nucleic acid molecule encoding a human MUM-2 protein. This invention provides an isolated nucleic acid molecule encoding a MUM protein operatively linked to a promoter of RNA transcription. This invention provides a vector comprising the an isolated cDNA encoding a MUM protein. This invention provides a vector which comprises an isolated cDNA encoding a MUM protein. This invention provides a vector which comprises an isolated cDNA encoding a MUM protein, wherein the vector is a plasmid. This invention provides a host cell for the vector which comprises an isolated cDNA encoding a MUM protein.

This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a MUM protein. This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM protein.

This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

This invention provides a nucleic acid probe comprising a the sequence of a nucleic acid molecule encoding a MUM-1 protein which is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14. This invention provides a nucleic acid probe comprising a the sequence of a nucleic acid molecule encoding a MUM-2 protein which is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14.

This invention provides a method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-1 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-1 protein. This invention provides a method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-2 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-2 protein.

This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-1 protein so as to prevent overexpression of the mRNA molecule. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-2 protein so as to prevent overexpression of the mRNA molecule.

This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an isolated cDNA molecule encoding a MUM protein. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the isolated genomic DNA molecule encoding a MUM protein. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an isolated RNA molecule encoding a MUM protein.

This invention provides a purified MUM protein. This invention provides a purified MUM-1 protein. This invention provides an antibody directed to a purified MUM-1 protein. This invention provides an antibody capable of specifically recognizing MUM-1 protein. This invention provides a purified MUM-2 protein. This invention provides an antibody directed to a purified MUM-2 protein. This invention provides an antibody capable of specifically recognizing a MUM-2 protein.

This invention provides a pharmaceutical compositior comprising an amount of an oligonucleotide effective to prevent overexpression of a human MUM-1 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane. This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide effective to prevent overexpression of a human MUM-2 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. JH-Cμ dissociation in BamHI digested DNA of the 14q+ SK-MM-1 cell line. A 10 μg of the high molecular weight DNA was completely digested with BamHI, loaded on each lane and blotted. The same filter was sequentially hybridized with JH, Cμ, Cγ2, and 0.7B/H probes. JH probe detects two rearranged bands of 12.0 kb and 9.7 kb. The 9.7 kb band is comigrated with that probed with Cγ2 probe, suggesting it to be a physiological rearrangement. On the other hand, one allele of the Cμ locus is deleted and another is rearranged (6.5 kb) without being comigrated with rearranged bands of JH. Therefore, 12.0 kb and 6.5 kb bands detected by JH and Cμ (shown by arrowheads) might represent unknown derivative chromosome and derivative 14 chromosome, respectively. As expected, 0.7B/H probe (FIG. 2A) detected the rearranged band comigrated with 6.5 kb band of Cμ. Dashed lines show the combination. Size markers of λ/HindIII are shown on the left.

FIGS. 2A-B. Molecular cloning of the breakpoints of the t(6;14) translocation and germline walking at MUM1 locus. (FIG. 2A) Restriction maps of λSKB-4a and λSKS-3 clones representing derivative 6 and 14 are shown, together with germline maps of IgH locus at 14q32 and MUM1 locus at 6p25. Arrows indicate the chromosomal breakpoints. B, BamHI; E, EcoRI; H, HindIII. (FIG. 2B) Comparison of the nucleotide sequences around the breakpoints on derivative 6 SEQ ID NO: 16 and derivative 14 SEQ ID NO: 17 chromosome. Homologous regions are indicated by dashes. The arrow indicates the breakpoint SEQ ID NO: 15. Nucleotide SEQ ID NO: 17 numbers shown below are the same as in the Sμ sequence reported by Sun, et al. (18).

FIGS. 3A-B. Mapping of the MUM1 locus to chromosome 6p25. (FIG. 3A) λMUM-3 genomic clone (FIG. 2A) was used as a probe for in situ hybridization. The white arrow indicates the fluorescence signal on chromosome 6 band p25. (FIG. 3B) Right panel shows the G-banding picture stained with DAPI.

FIGS. 4A-C. Expression of the MUM1 gene in hematopoietic lineage. A 10 μg aliquot of total RNA was loaded on each lane and Northern blot analysis was performed using the 2.1H probe (FIG. 2A). GAPDH or β-actin probes were used to control for amount of RNA loaded. (FIG. 4A) MUM1 RNA expression in various hematopoietic cell lines. MUM1 RNA is detected in B cell and mature T cell lines as a single 6 kb transcript. HELA, epithelial lineage; LCL, Epstein-Barr virus-transformed lymphoblastoid cell line; RAMOS and SK-MM-1, B-cell lineage; HUT-78 and MOLT-4, T-cell lineage; HL-60 and U937, myelomonocytic lineage; K562, erythroid lineage. Dashes indicate 28S and 18S. (FIG. 4B) Expression in B cell lines derived from various stages of B cell differentiation. MUM1 RNA is seen throughout the B cell development except for BJAB cell line. 697, pre-B cell stage; RAMOS and BJA-B, Burkitt cell line representing mature-B cell stage; RPMI-8226 and U-266, plasma cell stage. (FIG. 4C) Comparison of the expression level among myeloma cell lines. MUM1 RNA is overexpressed in SK-MM-1 cell line carrying t(6;14). Overexpression of the MUM1 is also demonstrated in XG-4, XG-7, and XG-10 cell lines. RPMI-8226, U-266, EJM, and SKMM-1 are IL-6 (interleukin-6) independent lines, whereas XG-1, XG-2, XG-4, XG-5, XG-6, XG-7, and XG-10 are IL-6 dependent lines.

FIGS. 5A-B1-B3. Sequence of MUM1 cDNA and structure of its predicted protein product. (FIG. 5A) Restriction map of the MUM1 cDNA and the position of the open reading frame (box). The solid box indicates approximate position of the DNA binding domain. Sc, SacII; A, ApaI; P, PstI; H, HindIII; S, SacI (FIGS. 5B1-5B3) Nucleotide sequence of the MUM1 cDNA SEQ ID NO: 13 and corresponding amino acid sequence SEQ ID NO: 14. Putative translation initiation codons and preceding stop codons appearing in frame are underlined. The asterisk indicates the translation stop codon.

FIGS. 6A-B. Homology between MUM1 SEQ ID NO: 1 and other IRF family proteins SEQ ID NOS: 2-7. (FIG. 6A) Similarity at N-terminal DNA binding domain. Black background indicates identical residues found more than four times. Gray indicates conserved residues that appear in at least four sequences at a given position. Conserved tryptophan residues in DNA binding domain among IRF family members are indicated by closed circles. (FIG. 6B) Similarity at C-terminal region between human MUM1 SEQ ID NO: 8, Mouse LSIRF/Pip SEQ ID NO: 9, Human ICSBP SEQ ID NO: 10, Human ISGF3λ SEQ ID NO: 11, and Human IRF-3 SEQ ID NO: 12. Black and gray background are as in (A).

FIG. 7. Genomic organization of the MUM1 gene and location of the chromosomal breakpoints in multiple myeloma. Filled boxes indicate the coding regions and empty boxes indicate the noncoding regions. The position, and the size of each exon of the HUM1 gene are approximate and have been determined by the hybridizations. One exon in each restriction fragment may consist of more than two exons. Translation initiation codon (ATG) and stop codon (TGA) are indicated. Genomic probes used for further investigations are shown as solid bars below the map. Arrows indicate the chromosomal breakpoints of SKMM-1 cell line and case 10. B, BamHI; E, EcoRI; H, HindIII.

FIG. 8. Scheme of the t(6;14) (p25;q32) translocation involving the MUM1 and the immunoglobulin heavy chain (IgH) gene loci. VH-D-J-CH indicates variable-diversity joining-constant region of the IgH gene. Direction of the MUM1 gene on the chromosome 6 is tentatively drawn.

FIGS. 9A-B. Demonstration of JH-Ca disjunction in U-266 cells and cloning of normal and 14q+ chromosomal breakpoints. (FIG. 9A) The panel shows the results of Southern blot analysis of BamHI digested U-266 and normal control (placenta) genomic DNA using the indicated JH and Cα probes. The arrowheads indicate two DNA fragments containing Cα sequences not linked to JH sequences, suggesting the presence of a chromosomal breakpoint in 14q32. (FIG. 9B) The panel provides a schematic representation of the phage clones isolated from a library constructed from U-266 DNA and screened with a Cα probe. Based on restriction enzyme analysis, the three cloned regions represent a normal Cα region (14q32 germ-line), and two rearranged regions (der.14 and 14q32) containing unknown sequences linked to Cα sequences. The 2.5BE probe used for Northern blot analysis of MUM2 transcripts (FIG. 10) is also shown.

FIG. 10. Identification of MUM2 RNA transcripts. The figure shows the results of a Northern blot analysis of RNA extracted from various MM/PCL cell lines using the 2.5BE probe (see FIG. 9) or GAPDH probe (as a control for RNA loading). A 1.9 Kb RNA transcript is detectable in some cell lines including U-266, indicating that the 2.5BE fragments represents part of a gene, MUM2.

FIGS. 11A-B. Schematic representation of IgH DNA rearrangements in normal B cells and in tumors carrying chromosomal translocations breaking the S region of the IgH locus. Note that in physiological IgH rearrangements (FIG. 11A) JH sequences and C sequences (Cμ before and Cγ after switch recombination, respectively) are consistently found within the same BamHI restriction fragment. Conversely, JH and C sequences are not linked, and are present on two different chromosomes [derivative X and derivative 14(14q+)] in cells carrying a chromosomal translocation breaking the switch region (FIG. 11B)

FIGS. 12A-B. MUM1 cDNA. cDNA insert is cloned into EcoRI/BamHI site of the pBluescript KS+. Bacteria strain used is DH5α cells. pcMUM1.16a contains full length open reading frame of nt. 217-1572.

FIG. 13. Breakpoint Cloning of the U-266 Cell Line. pMUM2-8 has a 22.0 KB insert in BamHI site of pBluescript KS+.

DETAILED DESCRIPTION OF THE INVENTION

The following standard abbreviations are used throughout the specification to indicate specific nucleotides:

C = cytosine A = adenosine T = thymidine G = guanosine

This invention provides a method of determining a chromosomal breakpoint in a subject suffering from multiple myeloma which comprises steps of: (a) obtaining a DNA sample from the subject suffering from multiple myeloma; (b) determining whether there is J and C disjunction in the immunoglobulin heavy chain gene in the obtained DNA sample; (c) obtaining a genomic library having clones which contain genomic DNA fragments from the DNA sample which shows positive J and C disjunction; (d) selecting and isolating clones of the obtained library which show positive hybridization with a probe which is capable of specifically hybridizing with the C but not the J region of the immunoglobulin heavy chain gene; (e) preparing fluorescent probes from the genomic DNA fragments of the isolated clones from step (d); (f) hybridizing said fluorescent probes with metaphase chromosomes; and (g) determining the identity of the chromosomes which are capable of hybridizing to said fluorescent probes, wherein the identification of a chromosome other than chromosome 14 would indicate that the chromosomal breakpoint is between chromosome 14 and the identified chromosome, thereby determining a chromosomal breakpoint in a subject suffering from multiple myeloma.

In an embodiment, step (b) of the above described method of this invention is performed by Southern blotting. In another embodiment, step (b) of the above method of this invention is performed by polymerase chain reaction (PCR) with appropriate probes. Polymerase chain reaction is well known in the art. Since the sequences of both the C and J regions of an immunoglobulin heavy chain gene are known, appropriate probes for PCR may routinely be designed.

In an embodiment, the genomic library is a phage vector library. In another embodiment, the genomic DNA fragments are generated by cleaving genomic DNA from cells of the subject with an appropriate restriction enzyme. In a further embodiment, the restriction enzyme is BamHI. In an embodiment, the restriction enzyme is Sau3AI. In another embodiment, the probe of step (d) is a human IgH J region JH probe. In a further embodiment, the probe of step (d) is a human IgH Cμ probe. In an embodiment, the probe of step (d) is a human IgH Cγ2 probe. In another embodiment, the chromosomal breakpoint identified is a t(6;14) (p25;q32) translocation. In an embodiment, the chromosomal breakpoint identified is a t(14;15) translocation.

This invention provides a method to identify a gene other than the immunoglobulin gene which is located in chromosome 14, altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma which comprises steps of: a) selecting a probe having a sequence of a chromosome other than chromosome 14, identified at the chromosomal breakpoint detected in a subject suffering from multiple myeloma, wherein said probe is capable of hybridizing to the unique sequence of the gene other than the immunoglobulin gene altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma; b) contacting said probe with mRNA isolated from a cell under conditions permitting formation of a complex between said probe and the mRNA; c) isolating the complex resulting from step (b); and d) determining the sequence of the mRNA in the isolated complex, thereby determining the identity of the gene.

In an embodiment, step (d) of the method to identify a gene other than the immunoglobulin gene which is located in chromosome 14, altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma comprises steps of: i) synthesizing complementary DNA to the mRNA; and ii) performing sequence analysis of the complementary DNA to determine the sequence of the mRNA.

This invention provides a gene identified by the method to identify a gene other than the immunoglobulin gene which is located in chromosome 14, altered by a chromosomal breakpoint detected in a subject suffering from multiple myeloma.

As used herein, “MUM” means any gene rearranged in 14q+ chromosomal abnormalities associated with multiple myeloma.

This invention provides a gene identified by the above method designated MUM-1. This invention provides a gene identified by the above method designated MUM-2.

This invention provides a gene identified by the above method, wherein the gene identified comprises a nucleic acid encoding a MUM protein. In an embodiment, the gene identified by the above method comprises a nucleic acid encoding a MUM-1 protein. In another embodiment, the gene identified by the above method comprises a nucleic acid encoding a MUM-2 protein.

This invention provides an isolated nucleic acid molecule encoding a MUM protein. In an embodiment, the isolated nucleic acid molecule encoding a MUM protein is a DNA molecule. In another embodiment, the isolated nucleic acid molecule encoding a MUM protein is a cDNA molecule.

In an embodiment, a cDNA nucleic acid molecule encoding a MUM-1 protein is cloned into a pBluescript KS+ and the resulting plasmid is designated as pcMUM1-1.6a (ATCC Accession No. 97579). Plasmid pcMUM1-1.6a was deposited on May 28, 1996 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Plasmid pcMUM1-1.6a was accorded ATCC Accession Number 97579.

In another embodiment, a partial cDNA nucleic acid molecule encoding a MUM-1 protein is cloned into a peluescript KS+ and the resulting plasmid is designated as pMUM1-2.4B/N (ATCC Accession No. 97578). Plasmid pMUM1-2.4B/N was deposited on May 28, 1996 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Plasmid pMUM1-2.4B/N was accorded ATCC Accession No. 97578.

In another embodiment, a partial cDNA nucleic acid molecule encoding a MUM-1 protein is cloned into a pBluescript KS+ and the resulting plasmid is designated as pMUM1-7.7B (ATCC Accession No. 97577). Plasmid pMUM1-7.7B was deposited or May 28, 1996 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Plasmid pMUM1-7.7B was accorded ATCC Accession Number 97577.

In another embodiment, a partial cDNA of the nucleic acid molecule encoding a MUM-2 protein is cloned into a pBluescript KS+ and the resulting plasmid is designated as pMUM2-8 (ATCC Accession No. 97580). Plasmid pMUM2-8 was deposited on May 28, 1996 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Plasmid pMUM2-8 was accorded ATCC Accession Number 97580.

In an embodiment, the isolated DNA molecule encoding a MUM protein is a cDNA molecule having the nucleotide sequence shown in FIG. 5B-3 (SEQ ID NO: 13).

In an embodiment, the isolated DNA molecule encoding a MUM protein is genomic DNA molecule. In an embodiment, the isolated nucleic acid molecule encoding a MUM protein is an RNA molecule.

In an embodiment, the isolated nucleic acid encodes a human MUM-1 protein. In another embodiment, the isolated nucleic acid molecule encodes a human MUM-2 protein.

In an embodiment, isolated nucleic molecule encodes the a human MUM-1 protein having substantially the same amino acid sequence as shown in FIG. 5B-2 (SEQ ID NO: 14). In an embodiment, isolated nucleic molecule encodes a human MUM-1 protein having the same amino acid sequence as shown in FIG. 5B (SEQ. ID NO). In an embodiment, the isolated nucleic acid molecule encoding a MUM protein is operatively linked to a promoter of RNA transcription.

This invention provides a vector comprising a cDNA molecule encoding a MUM protein. In an embodiment, a vector comprising cDNA encoding for MUM-1 is designated pcMUM1.6a. In an embodiment, a vector comprising partial cDNA encoding for MUM-1 is designated pMUM1.2.4B/N. In an embodiment, a vector comprising partial cDNA encoding for MUM-1 is designated pMUM1-7.7B. In an embodiment, a vector comprising partial cDNA encoding for MUM-2 is designated pMUM2-8. In an embodiment, a vector comprises genomic DNA encoding for MUM. In an embodiment, the vector is a plasmid. In an embodiment, a host cell comprises the vector comprising cDNA encoding for MUM. In an embodiment, a host cell comprises the vector comprising genomic DNA encoding for MUM. In a further embodiment, the host cell comprising vectors comprising cDNA encoding for MUM or comprising genomic DNA encoding for MUM is selected from a group consisting of a bacterial cell, a plant cell, and insect cell and a mammalian cell.

This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a MUM protein. This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM protein.

As used herein, the phrase “specifically hybridizing” means the ability of a nucleic acid molecule to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs.

In an embodiment, the nucleic acid probe specifically hybridizes with nucleic acid encoding MUM-1. In an embodiment, the nucleic acid probe is complementary to nucleic acid encoding MUM-1. In an embodiment, the nucleic acid probe specifically hybridizes with nucleic acid encoding MUM-2. In an embodiment, the nucleic acid probe is complementary to nucleic acid encoding MUM-2.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-1 is a DNA probe. In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-2 is a DNA probe.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-1 is a RNA probe. In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-2 is a RNA probe.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-1 is a genomic DNA probe. In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-2 is a genomic DNA probe.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-1 is labeled with a detectable marker. In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-2 is labeled with a detectable marker.

In an embodiment, the detectable marker is selected from the group consisting of a radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.

In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-1 is linked to a nucleic acid sequence capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule of human chromosome 14. In an embodiment, the nucleic acid probe which specifically hybridizes with nucleic acid encoding MUM-2 is linked to a nucleic acid sequence capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule of human chromosome 14.

This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the nucleic acid probe comprises a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM-1 protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the nucleic acid probe comprises a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM-2 protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the nucleic acid probe comprises a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM-1 protein which is linked at a specific break point to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the nucleic acid probe comprises a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a MUM-2 protein which is linked at a specific break point to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the specific break point of the nucleic acid probe comprises a portion of the t(6;14) (p25;q32) translocation. In an embodiment, the specific break point of the nucleic acid probe comprises a portion of a t(14;15) translocation. In an embodiment, the nucleic acid probe comprising a portion of the t(6;14) (p25;q32) translocation is labeled with a detectable marker. In an embodiment, the nucleic acid probe comprising a portion of a t(14;15) translocation is labeled with a detectable marker. In an embodiment, the nucleic acid probe comprising a portion of the t(6;14) (p25;q32) or comprising a portion of a t(14;15) translocation has a detectable marker selected from the group consisting of a radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.

This invention provides a method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-1 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-1 protein.

This invention provides a method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-2 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-2 protein.

In an embodiment, the rearrangement of nucleic acid encoding MUM-1 protein is detected by contacting the nucleic acid from the sample with a MUM-1 probe under conditions permitting the MUM-1 probe to hybridize with the nucleic acid encoding MUM-1 protein from the sample, thereby detecting the rearrangement of nucleic acid encoding MUM-1 protein in the sample.

In an embodiment, the rearrangement of nucleic acid encoding MUM-2 protein is detected by contacting the nucleic acid from the sample with a MUM-2 probe under conditions permitting the MUM-2 probe to hybridize with the nucleic acid encoding MUM-2 protein from the sample, thereby detecting the rearrangement of nucleic acid encoding MUM-2 protein in the sample.

In an embodiment, the rearrangement of nucleic acid encoding MUM-1 protein is detected by a MUM-1 probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-1 protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the rearrangement of nucleic acid encoding MUM-2 protein is detected by a the MUM-2 probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-2 protein which is linked to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 15.

In an embodiment, the MUM-1 probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-1 protein is linked at a specific break point to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 14.

In an embodiment, the MUM-2 probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding MUM-2 protein is linked at a specific break point to a nucleic acid sequence complementary to a sequence of a nucleic acid molecule of human chromosome 15.

In an embodiment, the MUM-1 probe comprises a specific break point comprising a portion of the t(6;14) (p25;q32) translocation. In an embodiment, the MUM-2 probe comprises a specific break point comprising a portion of a t(14;15) translocation.

In an embodiment, the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-1 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-1 protein comprises: a) obtaining DNA from the sample of the subject suffering from multiple myeloma; b) performing a restriction digest of the DNA with a panel of restriction enzymes; c) separating the resulting DNA fragments by size fractionation; d) contacting the resulting DNA fragments with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-1 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-1 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 and labeled with a detectable marker; e) detecting labeled bands which have hybridized to the nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-1 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-1 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 to create a unique band pattern specific to the DNA of subjects suffering from multiple myeloma; f) preparing DNA obtained from a sample of a subject for diagnosis by steps (a-e); and g) comparing the detected band pattern specific to the DNA obtained from a sample of subjects suffering from multiple myeloma from step (e) and the DNA obtained from a sample of the subject for diagnosis from step (f) to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.

In an embodiment, the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-2 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-2 protein comprises: a) obtaining DNA from the sample of the subject suffering from multiple myeloma; b) performing a restriction digest of the DNA with a panel of restriction enzymes; c) separating the resulting DNA fragments by size fractionation; d) contacting the resulting DNA fragments with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-2 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 and labeled with a detectable marker; e) detecting labeled bands which have hybridized to the nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-2 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 to create a unique band pattern specific to the DNA of subjects suffering from multiple myeloma; f) preparing DNA obtained from a sample of a subject for diagnosis by steps (a-e); and g) comparing the detected band pattern specific to the DNA obtained from a sample of subjects suffering from multiple myeloma from stee (e) and the DNA obtained from a sample of the subject for diagnosis from step (f) to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.

In an embodiment, the size fractionation in step (c) is effected by a polyacrylamide or agarose gel. In an embodiment, the detectable marker is radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.

In an embodiment, the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-1 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-1 protein comprises: a) obtaining RNA from the sample of the subject suffering from multiple myeloma; b) separating the RNA sample by size fractionation; c) contacting the resulting RNA species with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encodirg a human MUM-1 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-1 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 14 and labeled with a detectable marker; d) detecting labeled bands which have hybridized to the RNA species to create a unique band pattern specific to the RNA of subjects suffering from multiple myeloma; e) preparing RNA obtained from a sample of a subject for diagnosis by steps (a-d); and f) comparing the detected band patterr specific to the RNA obtained from a sample of subjecte suffering from multiple myeloma from step (d) and the RNA obtained from a sample of the subject for diagnosis from step (f) to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.

In an embodiment, the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-2 protein in a sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-2 protein comprises: a) obtaining RNA from the sample of the subject suffering from multiple myeloma; b) separating the RNA sample by size fractionation; c) contacting the resulting RNA species with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human MUM-2 protein, wherein the sequence of a nucleic acid molecule encoding a MUM-2 protein is linked at a specific break point to a specified nucleic acid sequence of human chromosome 15 and labeled with a detectable marker; d) detecting labeled bands which have hybridized to the RNA species to create a unique band pattern specific to the RNA of subjects suffering from multiple myeloma; e) preparing RNA obtained from a sample of a subject for diagnosis by steps (a-d); and f) comparing the detected band pattern specific to the RNA obtained from a sample of subjects suffering from multiple myeloma from step (d) and the RNA obtained from a sample of the subject for diagnosis from step (f) to determine whether the patterns are the same or different and to diagnose thereby predisposition to multiple myeloma if the patterns are the same.

In an embodiment, the size fractionation in step (b) is effected by a polyacrylamide or agarose gel. In an embodiment, the detectable marker is radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.

In an embodiment, multiple myeloma associated with the expression of a specific human MUM-1 is diagnosed by the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-1 protein in a DNA or RNA sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-1 protein.

In an embodiment, multiple myeloma associated with the expression of a specific human MUM-2 is diagnosed by the method for detecting a predisposition to multiple myeloma associated with the expression of a human MUM-12 protein in a DNA or RNA sample from a subject which comprises detecting in a sample from the subject a rearrangement of nucleic acid encoding MUM-2 protein.

This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-1 protein so as to prevent overexpression of the mRNA molecule. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-2 protein so as to prevent overexpression of the mRNA molecule.

This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the cDNA molecule encoding a MUM protein. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA molecule encoding a MUM protein. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the RNA molecule encoding a MUM protein.

This invention provides a purified MUM protein. This invention provides a purified MUM-1 protein. This invention provides a purified human MUM-1 protein. This invention provides an antibody directed to a purified MUM-1 protein. This invention provides an antibody capable of specifically recognizing MUM-1 protein. In an embodiment, the antibody capable of specifically recognizing MUM-1 protein is a human MUM-1 protein.

This invention provides a purified MUM-2 protein. This invention provides a purified human MUM-2 protein. This invention provides an antibody directed to a purified MUM-2 protein. This invention provides an antibody capable of specifically recognizing MUM-2 protein. In an embodiment, the antibody capable of specifically recognizing MUM-2 protein is a human MUM-2 protein.

In an embodiment, the antibody directed to a purified MUM-1 protein is a monoclonal antibody. In an embodiment, the antibody capable of specifically recognizing MUM-1 protein is a monoclonal antibody. In an embodiment, the antibody capable of specifically recognizing MUM-1 protein is a human MUM-1 protein.

In an embodiment, the antibody directed to a purified MUM-2 protein is a monoclonal antibody. In an embodiment, the antibody capable of specifically recognizing MUM-2 protein is a monoclonal antibody. In an embodiment, the antibody capable of specifically recognizing MUM-2 protein is a human MUM-2 protein.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-1 protein so as to prevent overexpression of the mRNA molecule effective to prevent overexpression of a human MUM-1 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a sequence capable of specifically hybridizing to a cDNA molecule encoding a MUM protein effective to prevent overexpression of a human MUM-1 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a sequence capable of specifically hybridizing to a genomic DNA molecule effective to prevent overexpression of a human MUM-1 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human MUM-2 protein so as to prevent overexpression of the mRNA molecule effective to prevent overexpression of a human MUM-2 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a sequence capable of specifically hybridizing to a cDNA molecule encoding a MUM protein effective to prevent overexpression of a human MUM-2 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide having a sequence capable of specifically hybridizing to a genomic DNA molecule effective to prevent overexpression of a human MUM-2 protein and a pharmaceutically acceptable carrier capable of passing through a cell membrane.

This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

EXPERIMENTAL DETAILS Materials and Methods

Cell Lines.

The following myeloma cell lines were used in the present study: SK-MM-l, RPMI-8226, U266, EJM, XG-1, XG-2, XG-4, XG-5, XG-6, XG-7, and XG-10. The RPMI-8226 cell line was obtained through the American Type Culture Collection (ATCC, Rockville, Md.). SKMM-1 and U-266 cell lines were gifts from Dr. A. N. Houghton and Dr. K. Nilsson, respectively (18; 12). Characterization of these cell lines were previously reported. Six XG cell lines were gifts from Dr. B. Klein and were cultured in RPMI 1640 containing 10% fetal calf serum (FCS), SxlO-smol/L 2-ME, and rIL-6(lng/mL)(13;19). Other myeloma cell lines used were all IL-6 independent. The SK-MM-l cell line was used to isolate the chromosomal breakpoint carrying the 14q+ chromosome without any information on the donor chromosome. XG-1, XG-2, XG-6, XG-8 cell lines are reported to carry the t(11;14)(q13;q32) translocation. XG-5 cells also share both t(11;14) and t(8;14)(q24;q32).

Southern and Northern Blot Analyses.

Southern blot analysis was performed as previously described (21). Briefly, ten micrograms of high molecular-weight DNA extracted from each cell line was digested to completion with BamHI and HindIII restriction enzymes, size-fractionated on 0.7% agarose gel, and transferred onto Duralose nitrocellulose membrane (Stratagene) according to the manufacturer's instructions. Blots were hybridized with a random-primed DNA probe and washed at 60° C. in 0.2×SSC and 0.1% SDS for 5 minutes. Genomic probes used in this study were as follows; human IgH J region JH probe (6.6 kb BamHI-HindIII fragment) was provided by Dr. J. V. Ravetch, human IgH Cμ probe (1.3 kb EcoRI fragment) was provided by Dr. S. J. Korsmeyer. Human IgH region Cγ2 probe was provided by Dr. C. Croce.

Northern blot analysis was performed as described previously (21). Briefly, a 10 μg aliquot of total RNA was loaded on each lane and probed with a 2.1H probe of the MUMI gene (FIG. 2A). GAPDH or β-actin probes were used as controls for amount of total RNA.

Genomic Library.

High molecular-weight DNA of SK-MM-1 cell line was digested completely with BamHI and partially with Sau3AI, and size-fractionated by using a low-melting point agarose gel. DNA ranging from 10 kb to 23 kb were purified and ligated into the BamHI sites of λ-DASH II phage vector (Stratagene, La Jolla, Calif.). After packaging, 3×10⁵ and 6×10⁵ recombinant clones of the BamHI digested library and partially digested library were screened with JH and Cμ probes, respectively. To isolate the germline region of the 6p25 locus, a commercially available human placental library (Stratagene) was screened. Positive clones were mapped with restriction enzymes by partial digestion of the phage DNAs followed by probing with T7 and T3 primers labeled with T4 polynucleokinase and 32p-γATP.

cDNA Library.

A phage library constructed by oligo-dT and random-priming normal human spleen RNA (Clontech) was screened by 2.1H probe (FIG. 2A) to isolate initial MUM1 cDNA clones. After the first round of screening, positive clones were used as probes to walk to the 5′ side using the same library. Positive clones were subcloned into pBluescript and analyzed for mapping and sequencing.

DNA Sequencing.

DNA sequences were determined by the dideoxy chain termination method and analyzed by an ABI(Applied Biosystems) autosequencer. Deletion mutants for sequencing were prepared using exonuclease III and mung bean nuclease. cDNA sequences were analyzed with the Genetics Computer Group (GCG) programs. Sequence homology searches were carried out through the BLAST E-mail server at the National Center for Biotechnology Information, National Library of Medicine, Bethesda, Md.

Fluorescence in situ Hybridization (FISH).

Metaphase chromosome from human lymphocytes were prepared. A biotin-labeled probe was prepared by nick-translation using Bio-16-dUTP. Conditions for hybridization and washing were described previously (22).

Experimental Results

IgH Gene Rearrangement of the SK-MM-I Cell Line.

In BamHI digestion, the JH probe detects two rearranged bands of the size of 12.0 kb and 9.7 kb (FIG. 1). The 9.7 kb band is comigrated with that probed with Cγ2 probe, suggesting it to be a physiological rearrangement, although this cell line secretes only λ chain. One allele of the Cμ locus is deleted and another is rearranged (6.5 kb) without being comigrated with rearranged bands of JH. Hybridization with a Cα probe showed only the germline band (data not shown). These results suggested the possibility of the chromosomal breakpoint between JH and Cμ locus. Hence, the 12.0 kb and the 6.5 kb bands detected by JH and Cμ were considered to represent unknown derivative chromosome and derivative 14 chromosome, respectively.

Molecular Cloning of the t(6;14) (p25;q32) Breakpoint.

A genomic library constructed with BamHI complete digestion was screened with a JH probe to isolate the 12.0 kb BamHI band. Another library constructed with Sau3AI partial digestion was screened with a Cμ probe to isolate phage clones containing the 6.5 kb BamHI fragment. Two phage clones, λSKB-4a and λ SKS-3, considered to represent the unknown derivative and derivative 14 chromosomes respectively, were obtained (FIG. 2A). A 0.7 kb BamHI-HindIII probe (0.7B/H) of the λSKS-3 was used to confirm the combination with the rearranged 6.5 kb Cμ band by Southern analysis (FIG. 1). The chromosomal origin of the centromeric side of the λSKB-4a and telomeric side of the λSKS-3 were confirmed by hybridization to a somatic cell hybrid DNA panel with a 4.5 kb ApaI fragment(4.5A) and 2.1 kb HindIII (2.1H) probes. Both probes showed positive signals in hybrid cell DNA containing a human chromosome 6 (data not shown). These probes were also used to isolate the germline chromosome 6 region by screening the human placental genomic library. One of the phage clone DNA (λMUM-3) was used as a probe for FISH analysis. It identified the localization of this region to be chromosome 6 short arm p25 (FIG. 3). To investigate the precise breakpoint within the IgH gene, a 1.5 kb HindIII-EcoRI fragment of the λSKS-3, containing the breakpoint on derivative 14 chromosome was sequenced. The breakpoint was confirmed to be just 3′ to the switch μ(S μ) repetitive sequences SEQ ID NO: 15 (FIG. 2B). Nucleotide sequencing of the region around the breakpoints of chromosome 6 and derivative 6 chromosome showed that the chromosomal translocation was reciprocal with minimum deletion of both the IgH and 6p25 sequences.

Transcriptional Unit in the Vicinity of the 6p25 Breakpoint.

An attempt to find a functional transcriptional unit in the vicinity of the breakpoints was made. Although a 4.5A probe on derivative 6 chromosome could not detect any transcripts, a 2.1H probe on derivative 14 chromosome detected a single 6 kb transcript in the SK-MM-1 cell line. Accordingly, this gene was designated as MUM1 (multiple myeloma oncogene 1). The same probe was used to study the expression of the MUM1 gene in various hematopoietic cell lines. The 6 kb message was expressed at high levels in most B cell lines and at low levels in peripheral T cell lines (FIG. 4A). Cell lines derived from immature T cells, the myelomonocytic lineage, and erythroid lineage do not seem to express MUM1. In B cells, MUM1 appears to be expressed throughout the development from the preB cell stage to the plasma cell stage (FIG. 4B). However, some of the Burkitt's lymphoma derived cell lines such as BJA-B did not express this gene (data not shown). The expression level of the MUM1 transcript in myeloma cell lines was also examined (FIG. 4C). The SK-MM-l cell line showed a 7.5-fold overexpression when compared with the other three IL-6 independent cell lines, suggesting a deregulated expression of the translocated allele. It is of interest that the IL-6 dependent XG-4, XG-7, and XG-10 cell lines are also expressing at high levels. Particularly, expression in the XG-7 cell line is 19.9 times the average of the aforementioned control cell lines.

MUMI cDNA Cloning, Sequencing, and Homology Search.

Human spleen cDNA library was initially screened with a 2.1H probe followed by three times walking to 5′ side using cDNA probes. A 5.5 kb cDNA, approximately corresponding to the size detected by Northern analysis was isolated. This cDNA contained a 1,353 base pair open reading frame (ORF) and a long 3′ untranslated region (FIG. 5A). The ORF encodes for a protein of 451 amino acids with a predicted molecular weight of 50 kD (FIG. 5B-1-5B-2 (SEQ ID NO: 14). The putative ATG initiation codon at position 217 has G at the −3 position which corresponds to the Kozak consensus sequence (23). The ORF is preceded by two in-frame stop codons. A database search demonstrated a significant similarity between MUM-1 ORF and the interferon regulatory factor (IRF) family proteins. The NH₂-terminal of the MUM-1 ORF (SEQ ID NO: 1) shares a high homology with all of the IRF family proteins (SEQ ID NOS: 2-7) which share a characteristic DNA binding motif consisting of the conserved 5 tryptophan residues (FIG. 6A). The COOH-terminal (SEQ ID NO: 8) also has a high homology with ICSBP (SEQ ID NO: 10) (interferon consensus sequence binding protein) (21), ISGF3γ (SEQ ID NO: 11) (interferon-stimulated gene factor-3 gamma) (22), and IRF-3 protein (SEQ ID NO: 12) (23) (FIG. 6B), although it did not have any homologous regions with IRF-1 and IRF-2 protein. The highest similarity (95.1%) and identity (SEQ ID NOS: 2 and 9) (91.8%) were found with a possible mouse homolog, LSIRF (lymphoid specific interferon regulatory factor)/Pip (PU-1 cofactor protein-1) (24,25). A high similarity was found with ICSBP (63.98%), ISGF3γ (55.8%), and IRF3 (50.1%) among the human IRF family protein members. A gene sequence encoding a nearly identical protein was recently deposited in GenBank. This gene, termed ICSAT (interferon consensus sequence binding protein in adult T-cell leukemia cell lines or activated T cells) is likely to be the same gene as MUM1 (26).

Breakpoints at MUM1 Locus in Multiple Myeloma.

In order to analyze the exact location of the SK-MM-l breakpoint at the 6p25 locus and to explore the frequency of the MUM1 gene involvement in myeloma cases, we walked nearly 55 kb in a human placental genomic phage library around the MUM1 gene and determined the rough exon-intron structure as shown in FIG. 7 (FIG. 7). The SK-MM-1 breakpoint was located 3′ to the last exon, containing a poly A additional signal, consistent with an unaltered size of the MUM1 transcript of this cell line in Northern analysis. Seven repeat-free genomic probes shown in FIG. 7 have been used to investigate the rearrangement in Southern analyses of the 11 MM cell lines and 18 MM cases. One case (case 10) displayed rearranged bands in BamHI and XbaI digests when analyzed using a 0.9A probe located at 3′ to the MUM1 gene.

Cloning of the MUM2 Locus from the U-266 Multiple Myeloma Cell Line.

Using an experimental strategy analogous to the one described for the cloning of the MUM1 gene from the SK-MM-1 cell line, a second genetic locus altered in multiple myeloma (MUM2) was identified by analyzing the U-266 multiple myeloma cell line. Briefly, Southern blot analysis using BamH restiction digestion and various Ig probes showed that U-266 DNA contained two rearranged fragments (shown by arrowheads in FIG. 9) containing Cα sequences and lacking J sequences. These two fragments (der 14 and 14q32 in FIG. 9) were cloned from a genomic library constructed from U-266 DNA along with a normal 14q 32 locus (14q32 germline in FIG. 9). In order to determine whether a gene was located in proximity to the chromosomal breakpoints in der 14, the 2.5 BE restriction fragment (see FIG. 9), which was at the opposite side of the Ig Ca sequences, was used to probe a Northern blot carrying RNA from various MM cell lines. The results (FIG. 10) showed that a 1.9 kb mRNA was detectable in some of these cell lines including U-266. This result showed that a gene, called MUM2, normally not present within the Ig locus on chromosome 14q32, had been translocated in proximity of the Ig locus in U-266 cells. Since the Ig locus contains strong transcriptional regulatory elements, it is likely that the expression of this gene is deregulated in these cells. The structure of the MUM2 gene and its protein are currently under investigation. The 2.5 BE probe and other probes derived from the der 14 phage can be used to screen MM cases for MUM2 rearrangements as shown for MUM1 (FIG. 7).

Experimental Discussion

Using the experimental strategies used for the identification of the MUM1 and MUM2 genes in the SK-MM-1 and U-266 cell lines, respectively, it is possible to analyze most MM cases and isolate the corresponding genes. The scheme shown in FIG. 11 shows that the physiological IgH gene rearrangements (FIG. 11A) typically maintain linkage of C and J sequences and this linkage becomes detectable by using an appropriate restriction enzyme digestion (BamHI in the example in FIG. 11). Conversely, chromosomal translocations (14q+) affecting the IgH locus on 14q32 lead to breakage of the C-J linkage and the two sets of sequences appear on distinct restriction fragments. (FIG. 11B) Table 1 shows the application of this analysis to a panel of MM cell lines and biopsies. The results show that at least 65% of cases show breakage of the C-J linkage within Ig J or switch regions. The restiction fragments containing either C or J sequences (R in Table 1) can be cloned as shown for the SK-MM-1 and U-266 cell lines and the genes flanking the chromosomal breakpoints can be used as probes to screen additional MM cases for similar rearrangements, whereas the sequence of the genes can be used to understand the consequences of these genetic lesions in multiple myeloma. Cloning of the chromosomal breakpoints and corresponding genes is currently ongoing for all of the MM cases shown in Table 1.

The method of analysis of 14q+ chromosomal translocations and identification of the genes altered in multiple myeloma of this invention will allow 1) the determination of chromosomal sequences involved in 14q+ translocations, the most important cytogenetic lesion associated with MM pathogenesis elucidation; 2) elucidation of specific gene lesions for MM; 3) a diagnostic method based on gene/DNA lesion and 4) a therapeutic approach aimed at counteracting the action of abnormal gene products.

TABLE 1 Summary of JH-C breakage analysis in MM cell lines and biopsies (cases). Rearrangement (R) involving physiologic Ig recombinations, i.e. retaining JH-C linkage are marked as R*; rearrangements lacking JH-C linkage, and therefore suggesting a 14q+ chromosomal breakpoint, are marked as R. The latter represents candidates for cloning an further analysis. possible Cell Line/Case sIg JH Cμ Cα Sγ3’ breakpoint locus RPMI-8226 λ D/D D/D G R /G Sγ U-266 Eλ R/D D/D R / R /G G Sα EJM Gλ R /R* D/D G R*/G JH˜Sμ XG-1 Aκ R*/D D/D R* G ND XG-2 Gλ R*/D D/D G R*/G ND XG-4 Gκ R*/ R D/D G R*/G JH˜Sμ XG-5 λ R /D D/D G G JH˜Sμ XG-6 Gλ R*/ R D/D G R*/G JH˜Sμ XG-7 Aκ R/D D/D R/G R/D Sγ XG-10 G R*/ R D/D G R*/G JH˜Sμ SK-MM-1 κ R*/ R R /D G R*/G JH˜Sμ CASE125 R* G G R* ND CASE33 R /R* G G R /R* Sγ CASE34 R* G R* G ND CASE93 R* G R* G ND CASE91 R* R* R G Sα CASE128 R* G R* G ND R*, comigrated bands with JH; R , target bands to isolate; ND, not determined Possible breakage in switch regions: Cell Lines 4/11 (36%) Cases 2/6 (33%) Total 6/17 (35%) Possible breakage in JH ˜ switch regions: Cell Lines 9/11 (82%) Cases 2/6 (33%) Total 11/17 (65%)

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20. Mazars, G-R., Portier, M., Zhang, X-G., Jourdan, M., Bataille, R., Theillet, C., Klein, B. Mutations of the pS3 gene in human myeloma cell lines. Oncogene 7: 1015-1018, 1992.

21. Iida, S., Seto M., Yamamoto, K., Tojo, A., Asano, S., Kamada, N., Ariyoshi, Y., Takahashi, T., Ueda, R. MLLT3 gene on 9p22 involved in t(9;11) leukemia encodes a serine/proline rich protein homologous to MLLT1 on 19p13. Oncogene 8(11): 3085-3095, 1993.

22. Rao, P. H., Murty, V. V. V. S., Gaidano, G., Hauptschein, R., Dalla-Favera, R., and Chaganti, R. S. K. Subregional mapping of 8 single copy loci to chromosome 6 by fluorescence in situ hybridization. Cytogenet. Cell Genet. 66:272-273, 1994.

23. Kozak, M. The scanning model for translation: an update. J. Cell. Biol. 108:229-241, 1989.

24. Driggers, P. H., Ennist, D. L., Gleason, S. L., Mak, W-H., Marka, M. S., Levi, B-Z., Flanagan, J. R., Appella, E., Ozato, K.: Proc Natl Acad Sci USA 87: 3743-3747, 1990.

25. Veals, S. A., Schindler, C., Leonardo, D., Fu, X-Y., Aebersold, R., Damell, J. E., Levy, D. E. Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and myb families of DNA-binding proteins. Mol Cell Biol 12: 3315-3324, 1992.

26. Grant, C. E., Vasa, M. Z., Deeley, R. G. cIRF-3, a new member of the interferon regulatory factor (IRF) family that is rapidly and transiently induced by dsRNA. Nucleic Acid Res 23:2137-2145, 1995.

27. Matsuyama, T., Grossman, A., Mittrucker, H-W., Siderovski, D. P., Kiefer, F., Kawakami, T., Richardson, C. D., Taniguchi, T., Yoshinaga, S. K., Mak, T. W. Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element(ISRE). Nucleic Acid Res 23:2127-2136, 1995.

28. Eisenbeis, C. F., Singh, H., Storb, U. Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes & Dev 9:1377-1387, 1995.

29. Yamagata, T., Nishida, J., Tanaka, T., Sakai, R., Mitani, K., Yoshida, M., Taniguchi, T., Yazaki, Y., Hirai, H. A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes.

Mol Cell Biol 16:1283-1294, 1996.

18 108 amino acids amino acid single linear peptide unknown 1 Lys Leu Arg Gln Trp Leu Ile Asp Gln Ile Asp Ser Gly Lys Tyr Pro 1 5 10 15 Gly Leu Val Trp Glu Asn Glu Glu Lys Ser Ile Phe Arg Ile Pro Trp 20 25 30 Lys His Ala Gly Lys Gln Asp Tyr Asn Arg Glu Glu Asp Ala Ala Leu 35 40 45 Phe Lys Ala Trp Ala Leu Phe Lys Gly Lys Phe Arg Glu Gly Ile Asp 50 55 60 Lys Pro Asp Pro Pro Thr Trp Lys Thr Arg Leu Arg Cys Ala Leu Asn 65 70 75 80 Lys Ser Asn Asp Phe Glu Glu Leu Val Glu Arg Ser Gln Leu Asp Ile 85 90 95 Ser Asp Pro Tyr Lys Val Tyr Arg Ile Val Pro Glu 100 105 108 amino acids amino acid single linear peptide unknown 2 Lys Leu Arg Gln Trp Leu Ile Asp Gln Ile Asp Ser Gly Lys Tyr Pro 1 5 10 15 Gly Leu Val Trp Glu Asn Glu Glu Lys Ser Val Phe Arg Ile Pro Trp 20 25 30 Lys His Ala Gly Lys Gln Asp Tyr Asn Arg Glu Glu Asp Ala Ala Leu 35 40 45 Phe Lys Ala Trp Ala Leu Phe Lys Gly Lys Phe Arg Glu Gly Ile Asp 50 55 60 Lys Pro Asp Pro Pro Thr Trp Lys Thr Arg Leu Arg Cys Ala Leu Asn 65 70 75 80 Lys Ser Asn Asp Phe Glu Glu Leu Val Glu Arg Ser Gln Leu Asp Ile 85 90 95 Ser Asp Pro Tyr Lys Val Tyr Arg Ile Val Pro Glu 100 105 108 amino acids amino acid single linear peptide unknown 3 Arg Met Arg Pro Trp Leu Glu Met Gln Ile Asn Ser Asn Gln Ile Pro 1 5 10 15 Gly Leu Ile Trp Ile Asn Lys Glu Glu Met Ile Phe Gln Ile Pro Trp 20 25 30 Lys His Ala Ala Lys His Gly Trp Asp Ile Asn Lys Asp Ala Cys Leu 35 40 45 Phe Arg Ser Trp Ala Ile His Thr Gly Arg Tyr Lys Ala Gly Glu Lys 50 55 60 Glu Pro Asp Pro Lys Thr Trp Lys Ala Asn Phe Arg Cys Ala Met Asn 65 70 75 80 Ser Leu Pro Asp Ile Glu Glu Val Lys Asp Gln Ser Arg Asn Lys Gly 85 90 95 Ser Ser Ala Val Arg Val Tyr Arg Met Leu Pro Pro 100 105 108 amino acids amino acid single linear peptide unknown 4 Arg Met Arg Pro Trp Leu Glu Glu Gln Ile Asn Ser Asn Thr Ile Pro 1 5 10 15 Gly Leu Lys Trp Leu Asn Lys Glu Lys Lys Ile Phe Gln Ile Pro Trp 20 25 30 Met His Ala Ala Arg His Gly Trp Asp Val Glu Lys Asp Ala Pro Leu 35 40 45 Phe Arg Asn Trp Ala Ile His Thr Gly Lys His Gln Pro Gly Val Asp 50 55 60 Lys Pro Asp Pro Lys Thr Trp Lys Ala Asn Phe Arg Cys Ala Met Asn 65 70 75 80 Ser Leu Pro Asp Ile Glu Glu Val Lys Asp Lys Ser Ile Lys Lys Gly 85 90 95 Asn Asn Ala Phe Arg Val Tyr Arg Met Leu Pro Leu 100 105 107 amino acids amino acid single linear peptide unknown 5 Arg Leu Arg Gln Trp Leu Ile Glu Gln Ile Asp Ser Ser Met Tyr Pro 1 5 10 15 Gly Leu Ile Trp Glu Asn Glu Glu Lys Ser Met Phe Arg Ile Pro Trp 20 25 30 Lys His Ala Gly Lys Gln Asp Tyr Asn Gln Glu Val Asp Ala Ser Ile 35 40 45 Phe Lys Ala Trp Ala Val Phe Lys Gly Lys Phe Lys Glu Gly Asp Lys 50 55 60 Ala Glu Pro Ala Thr Trp Lys Thr Arg Leu Arg Cys Ala Leu Asn Lys 65 70 75 80 Ser Pro Asp Phe Glu Glu Val Thr Asp Arg Ser Gln Leu Asp Ile Ser 85 90 95 Glu Pro Tyr Lys Val Tyr Arg Ile Val Pro Glu 100 105 107 amino acids amino acid single linear peptide unknown 6 Lys Leu Arg Asn Trp Val Val Glu Gln Val Glu Ser Gly Gln Phe Pro 1 5 10 15 Gly Val Cys Trp Asp Asp Thr Ala Lys Thr Met Phe Arg Ile Pro Trp 20 25 30 Lys His Ala Gly Lys Gln Asp Phe Arg Glu Asp Gln Asp Ala Ala Phe 35 40 45 Phe Lys Ala Trp Ala Ile Phe Lys Gly Lys Tyr Lys Glu Gly Asp Thr 50 55 60 Gly Gly Pro Ala Val Trp Lys Thr Arg Leu Arg Cys Ala Leu Asn Lys 65 70 75 80 Ser Ser Glu Phe Lys Glu Val Pro Glu Arg Gly Arg Met Asp Val Ala 85 90 95 Glu Pro Tyr Lys Val Tyr Gln Leu Leu Pro Pro 100 105 106 amino acids amino acid single linear peptide unknown 7 Arg Ile Leu Pro Trp Leu Val Ser Gln Leu Asp Leu Gly Gln Leu Glu 1 5 10 15 Gly Val Ala Trp Val Asn Lys Ser Arg Thr Arg Phe Arg Ile Pro Trp 20 25 30 Lys His Gly Leu Arg Gln Asp Ala Gln Gln Glu Asp Phe Gly Ile Phe 35 40 45 Gln Ala Trp Ala Glu Ala Thr Gly Ala Tyr Val Pro Gly Arg Asp Lys 50 55 60 Pro Asp Leu Pro Thr Trp Lys Arg Asn Phe Arg Ser Ala Leu Asn Arg 65 70 75 80 Lys Glu Gly Leu Arg Leu Ala Glu Asp Arg Ser Lys Asp Pro His Asp 85 90 95 Pro His Lys Ile Tyr Glu Phe Val Asn Ser 100 105 95 amino acids amino acid single linear peptide unknown 8 Lys Arg Leu Cys Gln Ser Thr Ile Tyr Trp Asp Gly Pro Leu Ala Leu 1 5 10 15 Cys Asn Asp Arg Pro Asn Lys Leu Glu Arg Asp Gln Thr Cys Lys Leu 20 25 30 Phe Asp Thr Gln Gln Phe Leu Ser Glu Leu Gln Ala Phe Ala His His 35 40 45 Gly Arg Ser Leu Pro Arg Phe Gln Val Thr Leu Cys Phe Gly Glu Glu 50 55 60 Phe Pro Asp Pro Gln Arg Gln Arg Lys Leu Ile Thr Ala His Val Glu 65 70 75 80 Pro Leu Leu Ala Arg Gln Leu Tyr Tyr Phe Ala Gln Gln Asn Ser 85 90 95 95 amino acids amino acid single linear peptide unknown 9 Lys Arg Leu Cys Gln Ser Arg Ile Tyr Trp Asp Gly Pro Leu Ala Leu 1 5 10 15 Cys Ser Asp Arg Pro Asn Lys Leu Glu Arg Asp Gln Thr Cys Lys Leu 20 25 30 Phe Asp Thr Gln Gln Phe Leu Ser Glu Leu Gln Val Phe Ala His His 35 40 45 Gly Arg Pro Ala Pro Arg Phe Gln Val Thr Leu Cys Phe Gly Glu Glu 50 55 60 Phe Pro Asp Pro Gln Arg Gln Arg Lys Leu Ile Thr Ala His Val Glu 65 70 75 80 Pro Leu Leu Ala Arg Gln Leu Tyr Tyr Phe Ala Gln Gln Asn Thr 85 90 95 96 amino acids amino acid single linear peptide unknown 10 Lys Arg Leu Cys Gln Gly Arg Val Phe Cys Ser Gly Asn Ala Val Val 1 5 10 15 Cys Lys Gly Arg Pro Asn Lys Leu Glu Arg Asp Glu Val Val Gln Val 20 25 30 Phe Asp Thr Ser Gln Phe Phe Arg Glu Leu Gln Gln Phe Tyr Asn Ser 35 40 45 Gln Gly Arg Leu Pro Asp Gly Arg Val Val Leu Cys Phe Gly Glu Glu 50 55 60 Phe Pro Asp Met Ala Pro Leu Arg Ser Lys Leu Ile Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Tyr Val Arg Gln Leu Ala Glu Glu Ala Gly Lys Ser Cys 85 90 95 96 amino acids amino acid single linear peptide unknown 11 Gln Arg Leu Cys Pro Ile Pro Ile Ser Trp Asn Ala Pro Gln Ala Pro 1 5 10 15 Pro Gly Pro Gly Pro His Leu Leu Pro Ser Asn Glu Cys Val Glu Leu 20 25 30 Phe Arg Thr Ala Tyr Phe Cys Arg Asp Leu Val Arg Tyr Phe Gln Gly 35 40 45 Leu Gly Pro Pro Pro Lys Phe Gln Val Thr Leu Asn Phe Trp Glu Glu 50 55 60 Ser His Gly Ser Ser His Thr Pro Gln Asn Leu Ile Thr Val Lys Met 65 70 75 80 Glu Gln Ala Phe Ala Arg Tyr Leu Leu Glu Gln Thr Pro Glu Gln Gln 85 90 95 100 amino acids amino acid single linear peptide unknown 12 Gln Arg Leu Gly His Cys His Thr Tyr Trp Ala Val Ser Glu Glu Leu 1 5 10 15 Leu Pro Asn Ser Gly His Gly Pro Asp Gly Glu Val Pro Lys Asp Lys 20 25 30 Glu Gly Gly Val Phe Asp Leu Gly Pro Phe Ile Val Asp Leu Ile Thr 35 40 45 Phe Thr Glu Gly Ser Gly Arg Ser Pro Arg Tyr Ala Leu Trp Phe Cys 50 55 60 Val Gly Glu Ser Trp Pro Gln Asp Gln Pro Trp Thr Lys Arg Leu Val 65 70 75 80 Met Val Lys Val Val Pro Thr Cys Leu Arg Ala Leu Val Glu Met Ala 85 90 95 Arg Val Gly Gly 100 5176 base pairs nucleic acid single linear other nucleic acid unknown CDS 217..1569 13 GCCTGACCAA CATGGTAAAA CCCCATCTCT GCTAAAACTA CAAAAAATTA GCTGGATGTG 60 GTGGCAGGGA ACCTGTCATC CCAGCTAGTT GGGAGACTGA GGCAGGAGAA TCGCTCGATC 120 TTGGGACCCA CCGCTGCCCT CAGCTCCGAG TCCAGGGCGA GTGCAGAGCA CAGCGGGCGG 180 AGGACCCCGG GCGCGGGCGC GGACGGCACG CGGGGC ATG AAC CTG GAG GGC GGC 234 Met Asn Leu Glu Gly Gly 1 5 GGC CGA GGC GGA GAG TTC GGC ATG AGC GCG GTG AGC TGC GGC AAC GGG 282 Gly Arg Gly Gly Glu Phe Gly Met Ser Ala Val Ser Cys Gly Asn Gly 10 15 20 AAG CTC CGC CAG TGG CTG ATC GAC CAG ATC GAC AGC GGC AAG TAC CCC 330 Lys Leu Arg Gln Trp Leu Ile Asp Gln Ile Asp Ser Gly Lys Tyr Pro 25 30 35 GGG CTG GTG TGG GAG AAC GAG GAG AAG AGC ATC TTC CGC ATC CCC TGG 378 Gly Leu Val Trp Glu Asn Glu Glu Lys Ser Ile Phe Arg Ile Pro Trp 40 45 50 AAG CAC GCG GGC AAG CAG GAC TAC AAC CGC GAG GAG GAC GCC GCG CTC 426 Lys His Ala Gly Lys Gln Asp Tyr Asn Arg Glu Glu Asp Ala Ala Leu 55 60 65 70 TTC AAG GCT TGG GCA CTG TTT AAA GGA AAG TTC CGA GAA GGC ATC GAC 474 Phe Lys Ala Trp Ala Leu Phe Lys Gly Lys Phe Arg Glu Gly Ile Asp 75 80 85 AAG CCG GAC CCT CCC ACC TGG AAG ACG CGC CTG CGG TGC GCT TTG AAC 522 Lys Pro Asp Pro Pro Thr Trp Lys Thr Arg Leu Arg Cys Ala Leu Asn 90 95 100 AAG AGC AAT GAC TTT GAG GAA CTG GTT GAG CGG AGC CAG CTG GAC ATC 570 Lys Ser Asn Asp Phe Glu Glu Leu Val Glu Arg Ser Gln Leu Asp Ile 105 110 115 TCA GAC CCG TAC AAA GTG TAC AGG ATT GTT CCT GAG GGA GCC AAA AAA 618 Ser Asp Pro Tyr Lys Val Tyr Arg Ile Val Pro Glu Gly Ala Lys Lys 120 125 130 GGA GCC AAG CAG CTC ACC CTG GAG GAC CCG CAG ATG TCC ATG AGC CAC 666 Gly Ala Lys Gln Leu Thr Leu Glu Asp Pro Gln Met Ser Met Ser His 135 140 145 150 CCC TAC ACC ATG ACA ACG CCT TAC CCT TCG CTC CCA GCC CAG CAG GTT 714 Pro Tyr Thr Met Thr Thr Pro Tyr Pro Ser Leu Pro Ala Gln Gln Val 155 160 165 CAC AAC TAC ATG ATG CCA CCC CTC GAC CGA AGC TGG AGG GAC TAC GTC 762 His Asn Tyr Met Met Pro Pro Leu Asp Arg Ser Trp Arg Asp Tyr Val 170 175 180 CCG GAT CAG CCA CAC CCG GAA ATC CCG TAC CAA TGT CCC ATG ACG TTT 810 Pro Asp Gln Pro His Pro Glu Ile Pro Tyr Gln Cys Pro Met Thr Phe 185 190 195 GGA CCC CGC GGC CAC CAC TGG CAA GGC CCA GCT TGT GAA AAT GGT TGC 858 Gly Pro Arg Gly His His Trp Gln Gly Pro Ala Cys Glu Asn Gly Cys 200 205 210 CAG GTG ACA GGA ACC TTT TAT GCT TGT GCC CCA CCT GAG TCC CAG GCT 906 Gln Val Thr Gly Thr Phe Tyr Ala Cys Ala Pro Pro Glu Ser Gln Ala 215 220 225 230 CCC GGA GTC CCC ACA GAG CCA AGC ATA AGG TCT GCC GAA GCC TTG GCG 954 Pro Gly Val Pro Thr Glu Pro Ser Ile Arg Ser Ala Glu Ala Leu Ala 235 240 245 TTC TCA GAC TGC CGG CTG CAC ATC TGC CTG TAC TAC CGG GAA ATC CTC 1002 Phe Ser Asp Cys Arg Leu His Ile Cys Leu Tyr Tyr Arg Glu Ile Leu 250 255 260 GTG AAG GAG CTG ACC ACG TCC AGC CCC GAG GGC TGC CGG ATC TCC CAT 1050 Val Lys Glu Leu Thr Thr Ser Ser Pro Glu Gly Cys Arg Ile Ser His 265 270 275 GGA CAT ACG TAT GAC GCC AGC AAC CTG GAC CAG GTC CTG TTC CCC TAC 1098 Gly His Thr Tyr Asp Ala Ser Asn Leu Asp Gln Val Leu Phe Pro Tyr 280 285 290 CCA GAG GAC AAT GGC CAC AGG AAA AAC ATT GAG AAC CTG CTG AGC CAC 1146 Pro Glu Asp Asn Gly His Arg Lys Asn Ile Glu Asn Leu Leu Ser His 295 300 305 310 CTG GAG AGG GGC GTG GTC CTC TGG ATG GCC CCC GAC GGG CTC TAT GCG 1194 Leu Glu Arg Gly Val Val Leu Trp Met Ala Pro Asp Gly Leu Tyr Ala 315 320 325 AAA AGA CTG TGC CAG AGC ACG ATC TAC TGG GAC GGG CCC CTG GCG CTG 1242 Lys Arg Leu Cys Gln Ser Thr Ile Tyr Trp Asp Gly Pro Leu Ala Leu 330 335 340 TGC AAC GAC CGG CCC AAC AAA CTG GAG AGA GAC CAG ACC TGC AAG CTC 1290 Cys Asn Asp Arg Pro Asn Lys Leu Glu Arg Asp Gln Thr Cys Lys Leu 345 350 355 TTT GAC ACA CAG CAG TTC TTG TCA GAG CTG CAA GCG TTT GCT CAC CAC 1338 Phe Asp Thr Gln Gln Phe Leu Ser Glu Leu Gln Ala Phe Ala His His 360 365 370 GGC CGC TCC CTG CCA AGA TTC CAG GTG ACT CTA TGC TTT GGA GAG GAG 1386 Gly Arg Ser Leu Pro Arg Phe Gln Val Thr Leu Cys Phe Gly Glu Glu 375 380 385 390 TTT CCA GAC CCT CAG AGG CAA AGA AAG CTC ATC ACA GCT CAC GTA GAA 1434 Phe Pro Asp Pro Gln Arg Gln Arg Lys Leu Ile Thr Ala His Val Glu 395 400 405 CCT CTG CTA GCC AGA CAA CTA TAT TAT TTT GCT CAA CAA AAC AGT GGA 1482 Pro Leu Leu Ala Arg Gln Leu Tyr Tyr Phe Ala Gln Gln Asn Ser Gly 410 415 420 CAT TTC CTG AGG GGC TAC GAT TTA CCA GAA CAC ATC AGC AAT CCA GAA 1530 His Phe Leu Arg Gly Tyr Asp Leu Pro Glu His Ile Ser Asn Pro Glu 425 430 435 GAT TAC CAC AGA TCT ATC CGC CAT TCC TCT ATT CAA GAA TGAAAAATGT 1579 Asp Tyr His Arg Ser Ile Arg His Ser Ser Ile Gln Glu 440 445 450 CAAGATGAGT GGTTTTCTTT TTCCTTTTTT TTTTTTTTTT TTTTGATACG GAGATACGGG 1639 GTCTTGCTCT GTCTCCCAGG CTGGAGTGCA GTGACACAAT CTCAGCTCAC TGTGACCTCC 1699 GCCTCCTGGG TTCAAGAGAC TCTCCTGCCT CAGCCTCCCT GGTAGCTGGG ATTACAGGTG 1759 TGAGCCACTG CACCCACCCA AGACAAGTGA TTTTCATTGT AAATATTTGA CTTTAGTGAA 1819 AGCGTCCAAT TGACTGCCCT CTTACTGTTT TGAGGAACTC AGAAGTGGAG ATTTCAGTTC 1879 AGCGGTTGAG GAGAATTGCG GCGAGACAAG CATGGAAAAT CAGTGACATC TGATTGGCAG 1939 ATGAGCTTAT TTCAAAAGGA AGGGTGGCTT TGCATTTTCT TGTGTTCTGT AGACTGCCAT 1999 CATTGATGAT CACTGTGAAA ATTGACCAAG TGATGTGTTT ACATTTACTG AAATGCGCTC 2059 TTTAATTTGT TGTAGATTAG GTCTTGCTGG AAGACAGAGA AAACTTGCCT TTCAGTATTG 2119 ACACTGACTA GAGTGATGAC TGCTTGTAGG TATGTCTGTG CCATTTCTCA GGGAAGTAAG 2179 ATGTAAATTG AAGAAGCCTC ACACGTAAAA GAAATGTATT AATGTATGTA GGAGCTGCAG 2239 TTCTTGTGGA AGACACTTGC TGAGTGAAGG AAATGAATCT TTGACTGAAG CCGTGCCTGT 2299 AGCCTTGGGG AGGCCCATCC CCCACCTGCC AGCGGTTTCC TGGTGTGGGT CCCTCTGCCC 2359 CACCCTCCTT CCCATTGGCT TTCTCTCCTT GGCCTTTCCT GGAAGCCAGT TAGTAAACTT 2419 CCTATTTTCT TGAGTCAAAA AACATGAGCG CTACTCTTGG ATGGGACATT TTTGTCTGTC 2479 CTACAATCTA GTAATGTCTA AGTAATGGTT AAGTTTTCTT GTTTCTGCAT CTTTTTGACC 2539 CTCATTCTTT AGAGATGCTA AAATTCTTCG CATAAAGAAG AAGAAATTAA GGAACATAAA 2599 TCTTAATACT TGAACTGTTG CCCTTCTGTC CAAGTACTTA ACTATCTGTT CCCTTCCTCT 2659 GTGCCACGCT CCTCTGTTTG TTTGGCTGTC CAGCGATCAG CCATGGCGAC ACTAAAGGAG 2719 GAGGAGCCGG GGACTCCCAG GCTGGAGAGC ACTGCCAGGA CCCACCACTG GAAGCAGGAT 2779 GGAGCTGACT ACGGAACTGC ACACTCAGTG GGCTGTTTCT GCTTATTTCA TCTGTTCTAT 2839 GCTTCCTCGT GCCAATTATA GTTTGACAGG GCCTTAAAAT TACTTGGCTT TTTCCAAATG 2899 CTTCTATTTA TAGAAATCCC AAAGACCTCC ACTTGCTTAA GTATACCTAT CACTTACATT 2959 TTTGTGGTTT TGAGAAAGTA CAGCAGTAGA CTGGGGCGTC ACCTCCAGGC CGTTTCTCAT 3019 ACTACAGGAT ATTTACTATT ACTCCCAGGA TTCAGCAGAA GATTGCGTTA GCTCTCAAAT 3079 GTGTGTTCCT GCTTTTCTAA TGGATATTTT AAATTCATTC AACAAGCACC TAGTAAGTGC 3139 CTGCTGTATC CCTACATTAC ACAGTTCAGC CTTTATCAAG CTTAGTGAGC AGTGAGCACT 3199 GAAACATTAT TTTTTAATGT TTAAAAAGTT TCTAATATTA AAGTCAGAAT ATTAATACAA 3259 TTAATATTAA TATTAACTAC AGAAAAGACA AACAGTAGAG AACAGCAAAA AAATAAAAAG 3319 GATCTCCTTT TTTCCCAGCC CAAATTCTCC TCTCTAAAAG TGTCCACAAG AAGGGGTGTT 3379 TATTCTTCCA ACACATTTCA CTTTTCTGTA AATATACATA AACTTAAAAA GAAAACCTCA 3439 TGGAGTCATC TTGCACACAC TTTTCATGCA GTGCTCTTTG TAGCTAAACA GTGAAGATTT 3499 ACCTCGTTCT GCTCAGAGGC CTTGCTGTGG AGCTCCACTG CCATGTACCC AGTAGGGTTT 3559 GACATTTCAT TAGCCATGCA ACATGGATAT GTATTGGGCA GCAGACTGTG TTTCGTGAAC 3619 TGCAGTGATG TATACATCTT ATAGATGCAA AGTATTTTGG GGTATATTAT CCTAAGGGAA 3679 GATAAAGATG ATATTAAGAA CTGCTGTTTC ACGGGGCCCT TACCTGTGAC CCTCTTTGCT 3739 GAAGAATATT TAACCCCACA CAGCACTTCA AAGAAGCTGT CTTGGAAGTC TGTCTCAGGA 3799 GCACCCTGTC TTCTTAATTC TCCAAGCGGA TGCTCCATTT CAATTGCTTT GTGACTTCTT 3859 CTTCTTTGTT TTTTTAAATA TTATGCTGCT TTAACAGTGG AGCTGAATTT TCTGGAAAAT 3919 GCTTCTTGGC TGGGGCCACT ACCTCCTTTC CTATCTTTAC ATCTATGTGT ATGTTGACTT 3979 TTTAAAATTC TGAGTGATCC AGGGTATGAC CTAGGGAATG AACTAGCTAT GGAAATAACT 4039 CAGGGTTAGG AATCCTAGCA CTTGTCTCAG GACTCTGAAA AGGAACGGCT TCCTCATTCC 4099 TTGTCTTGAT AAAGTGGAAT TGGCAAACTA GAATTTAGTT TGTACTCAGT GGACAGTGCT 4159 GTTGAAGATT TGAGGACTTG TTAAAGAGCA CTGGGTCATA TGGAAAAAAT GTATGTGTCT 4219 CCCCAGGTGC ATTTTCTTGG TTTATGTCTT GTTCTTGAGA TTTTGTATAT TTAGGAAAAC 4279 CTCAAGCAGT AATTAATATC TCCTGGAACA CTATAGAGAA CCAAGTGACC GACTCATTTA 4339 CAACTGAAAC CTAGGAAGCC CCTGAGTCCT GAGCGAAAAC AGGAGAGTTA GTCGCCCTAC 4399 AGAAAACCCA GCTAGACTAT TGGGTATGAA CTAAAAAGAG ACTGTGCCAT GGTGAGAAAA 4459 ATGTAAAATC CTACAGTGGA ATGAGCAGCC CTTACAGTGT TGTTACCACC AAGGGCAGGT 4519 AGGTATTAGT GTTTGAAAAA GCTGGTCTTT GAGCGAGGGC ATAAATACAG CTAGCCCCAG 4579 GGGTGGAACA ACTGTGGGAG TCTTGGGTAC TCGCACCTCT TGGCTTTGTT GATGCTCCGC 4639 CAGGAAGGCC ACTTGTGTGT GCGTGTCAGT TACTTTTTTA GTAACAATTC AGATCCAGTG 4699 TAAACTTCCG TTCATTGCTC TCCAGTCACA TGCCCCCACT TCCCCACAGG TGAAAGTTTT 4759 TCTGAAGTGT TGGGATTGGT TAAGGTCTTT ATTTGTATTA CGTATCTCCC CAAGTCCTCT 4819 GTGGCCAGCT GCATCTGTCT GAATGGTGCG TGAAGGCTCT CAGACCTTAC ACACCATTTT 4879 GTAAGTTATG TTTTACATGC CCCGTTTTTG AGACTGATCT CGATGCAGGT GGATCTCCTT 4939 GAGATCCTGA TAGCCTGTTA CAGGAATGAA GTAAAGGTCA GTTTTTTTTG TATTGATTTT 4999 CACAGCTTTG AGGAACATGC ATAAGAAATG TAGCTGAAGT AGAGGGGACG TGAGAGAAGG 5059 GCCAGGCCGG CAGGCCAACC CTCCTCCAAT GGAAATTCCC GTGTTGCTTC AAACTGAGAC 5119 AGATGGGACT TAACAGGCAA TGGGGTCCAC TTCCCCCTCT TCAGCATCCC CCGTACC 5176 451 amino acids amino acid linear protein unknown 14 Met Asn Leu Glu Gly Gly Gly Arg Gly Gly Glu Phe Gly Met Ser Ala 1 5 10 15 Val Ser Cys Gly Asn Gly Lys Leu Arg Gln Trp Leu Ile Asp Gln Ile 20 25 30 Asp Ser Gly Lys Tyr Pro Gly Leu Val Trp Glu Asn Glu Glu Lys Ser 35 40 45 Ile Phe Arg Ile Pro Trp Lys His Ala Gly Lys Gln Asp Tyr Asn Arg 50 55 60 Glu Glu Asp Ala Ala Leu Phe Lys Ala Trp Ala Leu Phe Lys Gly Lys 65 70 75 80 Phe Arg Glu Gly Ile Asp Lys Pro Asp Pro Pro Thr Trp Lys Thr Arg 85 90 95 Leu Arg Cys Ala Leu Asn Lys Ser Asn Asp Phe Glu Glu Leu Val Glu 100 105 110 Arg Ser Gln Leu Asp Ile Ser Asp Pro Tyr Lys Val Tyr Arg Ile Val 115 120 125 Pro Glu Gly Ala Lys Lys Gly Ala Lys Gln Leu Thr Leu Glu Asp Pro 130 135 140 Gln Met Ser Met Ser His Pro Tyr Thr Met Thr Thr Pro Tyr Pro Ser 145 150 155 160 Leu Pro Ala Gln Gln Val His Asn Tyr Met Met Pro Pro Leu Asp Arg 165 170 175 Ser Trp Arg Asp Tyr Val Pro Asp Gln Pro His Pro Glu Ile Pro Tyr 180 185 190 Gln Cys Pro Met Thr Phe Gly Pro Arg Gly His His Trp Gln Gly Pro 195 200 205 Ala Cys Glu Asn Gly Cys Gln Val Thr Gly Thr Phe Tyr Ala Cys Ala 210 215 220 Pro Pro Glu Ser Gln Ala Pro Gly Val Pro Thr Glu Pro Ser Ile Arg 225 230 235 240 Ser Ala Glu Ala Leu Ala Phe Ser Asp Cys Arg Leu His Ile Cys Leu 245 250 255 Tyr Tyr Arg Glu Ile Leu Val Lys Glu Leu Thr Thr Ser Ser Pro Glu 260 265 270 Gly Cys Arg Ile Ser His Gly His Thr Tyr Asp Ala Ser Asn Leu Asp 275 280 285 Gln Val Leu Phe Pro Tyr Pro Glu Asp Asn Gly His Arg Lys Asn Ile 290 295 300 Glu Asn Leu Leu Ser His Leu Glu Arg Gly Val Val Leu Trp Met Ala 305 310 315 320 Pro Asp Gly Leu Tyr Ala Lys Arg Leu Cys Gln Ser Thr Ile Tyr Trp 325 330 335 Asp Gly Pro Leu Ala Leu Cys Asn Asp Arg Pro Asn Lys Leu Glu Arg 340 345 350 Asp Gln Thr Cys Lys Leu Phe Asp Thr Gln Gln Phe Leu Ser Glu Leu 355 360 365 Gln Ala Phe Ala His His Gly Arg Ser Leu Pro Arg Phe Gln Val Thr 370 375 380 Leu Cys Phe Gly Glu Glu Phe Pro Asp Pro Gln Arg Gln Arg Lys Leu 385 390 395 400 Ile Thr Ala His Val Glu Pro Leu Leu Ala Arg Gln Leu Tyr Tyr Phe 405 410 415 Ala Gln Gln Asn Ser Gly His Phe Leu Arg Gly Tyr Asp Leu Pro Glu 420 425 430 His Ile Ser Asn Pro Glu Asp Tyr His Arg Ser Ile Arg His Ser Ser 435 440 445 Ile Gln Glu 450 154 base pairs nucleic acid single linear DNA (genomic) unknown 15 TTTTCTCTCT ACAGTCACCT CCCTGTTTAC CAAAGATAAT CACAATAAGT CCAGTTTACT 60 TACAAAACAA GTTTAGTTAT TAGAGGAAAC TAAAACTTCA GGATTCAGTC CAGATAATTT 120 TTAAAAACTC TAAAACAATG GACAGGGCTA GAAT 154 75 base pairs nucleic acid single linear DNA (genomic) unknown 16 TGGGCTCGGC CTGGTGGGGC AGCCACAGCG GGACGCAGTA GTGAAAGTCC AGTTTACTTA 60 CAAAACAAGT TTAGT 75 122 base pairs nucleic acid single linear DNA (genomic) unknown 17 TGGGCTCGGC CTTGGTGGGG CAGCCACAGC GGGACGCAAG TAGTGAGGGC ACTCAGAACG 60 CCACTCAGCC CCGACAGGCA GGGCACGAGG AGGCAGCTCC TCACCCTCCC TTTCTCTTTT 120 GT 122 77 base pairs nucleic acid single linear DNA (genomic) unknown 18 TATTAGAGGA AACTAAAACT TCAGGATTCA GCAGGGCATG AGGAGGCAGC TCCTCACCCT 60 CCCTTTCTCT TTTGTAC 77 

What is claimed is:
 1. An isolated nucleic acid molecule encoding a MUM-1 protein having the amino acid sequence shown in FIGS. 5B-1-5B-3 (SEQ. ID NO 14).
 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule is a DNA molecule.
 3. The isolated DNA molecule of claim 2, wherein the DNA molecule is a cDNA molecule.
 4. The isolated DNA molecule of claim 3, wherein the DNA molecule is a cDNA molecule having the nucleotide sequence shown in FIG. 5B (SEQ. ID NO 13).
 5. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule is an RNA molecule.
 6. The isolated nucleic acid molecule of claim 1 operatively linked to a promoter of RNA transcription.
 7. A vector comprising the nucleic acid molecule of claim
 1. 8. A vector comprising the nucleic acid molecule of claim
 3. 9. The vector of claim 8, wherein the vector is a plasmid.
 10. The plasmid of claim 9, designated pcMUM1-1.6a (ATCC Accession No. 97579).
 11. The plasmid of claim 9, designated pMUM1-2.4B/N (ATCC Accession No. 97578).
 12. The plasmid of claim 9, designated pMUM1-7.7B (ATCC Accession No. 97577).
 13. A host cell comprising the vector of claims 7, 8 or
 9. 14. The host cell of claim 13, wherein the cell is selected from the group consisting of a bacterial cell; a plant cell; an insect cell; and a mammalian cell. 